Bl21 ai escherichia coli

The Snr1 open reading frame (cDNA clone GH08712, DGRC) was cloned into pENTR-D (Thermo Fisher) and transferred to pDEST-17 (Invitrogen). BL21-AI Escherichia coli (Thermo Fisher C6070-03) were transformed with plasmid DNA and grown to an OD₆₀₀ of 0.4 at 37°C. Protein expression was induced with 0.2% L-arabinose (Sigma) at 25°C for 3 h. The cell pellet was lysed by incubation in 8 M urea (Sigma) in 1×PBS and a cleared lysate prepared and applied to a 1 ml HisTrap column (Cytavia 17524701), using a AKTA-Start Purification System. Purified protein was eluted with a stepwise imidazole gradient. Fractions containing purified Snr1 were pooled and desalted overnight by buffer exchange, concentrated to 1 μg/μl using Amicon Ultra 15 centrifugal filter unit (MilliporeSigma UFC901008 MWCO 10 Da) and injected into rabbits by Pocono Rabbit Farms and Laboratories (Canadensis, PA, USA). Affinity purification was performed by cloning Snr1 into pDEST-15 (Invitrogen) to express GST-tagged Snr1, which was cross linked to Glutathione Sepharose 4B beads (Cytavia 17075601) with 100 mM Dimethyl Pimelimidate (Sigma D8388). Antibody was validated by immunofluorescence and western blot on Drosophila larval brain (Fig. S1A,B).

Recombinant Src was expressed in BL21-AI Escherichia coli (Thermo) containing the pEX-Src-C-His (Origene, Blue Heron Biotech) and purified in a modified procedure as previously described [30 (link)]. The Src kinase assay used was the ADP-GLO assay (Promega) according to manufacturer protocol. Specifically, the kinase (1 ng) was incubated with between 10 nm-10 mM JVG045 compound/AZD0530 in 15 μL of kinase buffer for 30 min prior to the addition of 10 μL substrate solution containing ATP and poly[4Glu:Tyr] (Sigma). This reaction was allowed to react at RT for 1 h prior to quenching with ADP-Glo reagent for 40 min, followed by the addition of ADP-GLO detection reagent for 30 min prior to reading luminescence on a 96-well microplate reader.

The vectors were transformed into BL21-AI Escherichia coli (Thermo Fisher Scientific) and grown in supplemented super broth medium overnight at 25°C with autoinduction as previously described (29 (link)). Cells were lysed using a lysis buffer and a freeze-thaw cycle, followed by subsequent purification of the protein using His Mag Sepharose Ni beads (Cytivia, formerly GE Healthcare). After elution, the protein eluates were immediately adjusted to pH 5 by the addition of an MES buffer and analyzed for purity by SDS–polyacrylamide gel electrophoresis (fig. S1A) and concentration by NanoDrop (Thermo Fisher Scientific).

The cloned constructs were transformed into BL21-AI Escherichia coli (Thermo Fisher Scientific, Waltham, USA), and the NAGs were expressed and purified as described (26 (link)), using a modified elution buffer with a pH of 2.0. The eluted and equilibrated NAGs were analyzed for purity by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) as described, and their concentration was measured using a nanophotometer (Implen, Munich, Germany).